All human disease develops as a result from alterations in genetic or environmental factors or the combination of both. Cardiovascular diseases, which are the world's leading cause of death, are the best examples. In this disease genetic and environmental factors “join” to induce the endothelial pathology leading to cardiovascular and heart disease. Systemic and local delivery of genes or gene expression modifying agents could serve as the future arm of therapy. To achieve this, a temporary alteration to a particular layer of tissue below the surface of tissue, which forms pores without perturbing the overlying tissue, is needed for the facilitated entry of genetic material.
Some genetically based diseases result in disease states that can be retarded even without the addition of genetic material. They require alteration and diagnosis of a specific component below the surface of the tissue without perturbing the overlying tissue. The nature of the required alteration can extend from permanent destruction through a bleaching of a certain component of the tissue. An example of such a disease is called age related macular degeneration (AMD) in which a pigment that accumulates behind the retina has to be bleached under conditions that do not touch the overlying retinal tissue.
There are also infections that require a total destruction of tissue without affecting the overlying and underlying tissue. An example is a fungal infection in an underlying tissue layer that must be removed without damaging the overlying tissue. A specific case is fungal infection under cutaneous tissue like the nail of the foot that has to be destroyed without damaging the overlying or underlying tissue.
All of these problems have been incredibly difficult to address with existing technology. For example, successful solutions to these problems require effective calibration with a defined, in vivo, methodology for depth of penetration and the exact parameters required for minimal collateral damage. These parameters have to be checked with a defined diagnostic procedure, other than standard pathological techniques that are filled with artifacts from the fixation procedure. Thus, standard pathology is incapable of defining, with the sensitivity required, the parameters for highly controlled treatment.
Laser methods have been applied in the past to each of the problems previously described, but with limited success. All of these previous applications have used a linear form of laser tissue interaction, which cannot highlight a specific tissue layer selectively without an injection of an external highlighting light absorbing agent, which is the case for palliative laser attempts for AMD progress retardation using photodynamic therapy (see H. Sun and J. Nathans, “The Challenge of Macular Degeneration,” Scientific American, October 2001, p 61). Except for such protocols, in which injection of a highlighting absorbing substance is required, all other laser methodologies that have been applied to these problems have an effect of the laser that is limited to the surface. Alternatively, these methodologies require transport of the laser beam to a highly specific area, in a highly limiting fashion, by way of an invasive intrusion, for example with an optical fiber or other laser guiding device.
Thus, for example, no previous solutions of AMD have been able to bleach the subretinal pigments that are the cause of this disease without collateral damage to the overlying retina. This is the case even though higher order laser effects are known in microscopic analysis and can interrogate specific layers with the characteristics required (see analysis of T. Wilson and C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy Academic Press, New York 1984). These effects could not be effectively be used in therapy without the controlled in vivo characterization of parameters required for ultralow to zero collateral damage to the surrounding tissue. Thus, no such treatments have even been considered because of these problems (see H. Sun and J. Nathans, “The Challenge of Macular Degeneration,” Scientific American, October 2001, p 61).
In addition to AMD, fungal infections have remained essentially impossible to eradicate in places like the region under the nails of the feet, because of the lack of accurate parameterization for the highly specific, highly controlled treatments that are required.
Furthermore, no previous invention or report had shown site specific, prolonged expression of genetic material administration in vivo with any type of laser-related methodology (see for example Tao et al PNAS (USA) 84:4180-4, 1987; Kurata and Ikawa Cell Struct Funct 11:205-7, 1986; Paulombo et al J Photochem Photobiol 36:41-6, 1996).
Laser-related methodologies have been disclosed for example in U.S. Pat. No. 6,251,099, which teaches the use of pulsed laser light in order to generate “impulse transients” for delivering substances through the skin. These impulse transients generate transient increases in the permeability of epithelial tissue, thereby enabling the substances to penetrate. However, the laser light is not described as being useful for administering substances and/or performing therapeutic treatments within intermediate tissue layers, as would be required for the treatment of AMD, for example.
U.S. Pat. No. 4,775,361 discloses a method for administering a therapeutic substance through the skin of a patient, by using a pulsed laser beam of controlled wavelength, pulse length, pulse energy, pulse number and pulse repetition rate, sufficient to ablate the stratum corneum (outer layer of the skin) without damaging the epidermis. The therapeutic substance is then applied to the area of skin with the ablated stratum corneum. However, the disclosure still requires destruction of a portion of tissue. Therefore, the disclosed device of U.S. Pat. No. 4,775,361 could not be used for treatment of AMD, as it would damage retinal tissue above the area to be treated.
U.S. Pat. No. 5,713,845 describes the use of laser to force drugs into the skin, for example on small graphite particles which act as an explosive absorber of light energy. The laser beam is transmitted in very short pulses, which cause small explosions that force the drug through the skin. Clearly, the disclosed system is not suitable for applications in which the laser has to penetrate some distance of tissue before reaching the tissue to be treated. Thus, the disclosed system could not be used to treat AMD, as it would also damage retinal tissue above the area to be treated.
Gene therapy itself faces many obstacles before it will become a widely available method of treatment. A major obstacle in applying theoretical and experimental gene therapy methods into clinical practice is the current complexity of gene delivery systems. Viral vectors for gene delivery have shown great promise in relation to their efficiency, longevity and targeting capacities (1). The use of retroviral vectors for gene delivery in correcting genetic maladies in children was implemented clinically and initially, showed promise (2, 3). However, a number of unresolved issues concerning viral gene delivery remain. These include, among others: the potential for anti-viral immunological reactions; risk for development of malignant phenotypes associated with improper gene integration; size limitations on vector capacity; and challenges in the production of Good Manufacturing Practice (GMP) grade genetic material free of replication-competent viruses, that is suitable for clinical use (4). Hence, attention has focused on the use of non-viral methods of gene delivery such as cationic liposomes that transport foreign genes through cell membranes, or “naked DNA” constructs in which the desired gene is incorporated into a plasmid that may be injected directly into muscle or other tissues (5, 6). This latter technique requires physical methods such as electroporation (EP), that transiently fenestrate the cellular and nuclear membranes (5, 6). However, the in vivo efficiency of these methods is often low. Recent modifications such as the use of ultrasound energy (7) or microfabricated devices (8) to enhance naked DNA uptake in muscle or the dermis, respectively, have been successful in specific cases. Other potentially powerful genetic therapy tools include: anti-sense nucleotides, ribozymes, intron I and II based nucleic acids, and therapeutic small interference RNA (RNAi), some of which have been assessed in animal models and in preliminary clinical trials (9, 10). However, most methods still face significant obstacles in their specific applications due to gene delivery problems. One of these is that in vivo electroporation of naked DNA into large animals, even with enhancing delivery molecules such as polyethylenimine, will likely require a high-energy pulse>500 V (11) that while theoretically efficient for gene transduction, would not be practical as it would create considerable risk for local tissue injury (burn) or other deleterious effects (cardiac arrest).